Use of parvovirus for brain tumor therapy

ABSTRACT

Treatment of brain tumors by a composition including parvovirus, e.g., parvovirus H1, LuIII, Mouse minute virus (MMV), Mouse parvovirus (MPV), Rat minute virus (RMV), Rat parvovirus (RPV), Rat virus (RV), vectors based on the foregoing viral species, and/or cells capable of actively producing the foregoing viral species. Also described is the use of a parvovirus for the preparation of a pharmaceutical composition for the treatment of a brain tumor. Tumors for which the compositions and methods of the invention have particular utility include glioma, medulloblastoma and meningioma.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the use of a parvovirus,preferably parvovirus H1 or a related parvovirus such as LuIII, Mouseminute virus (MMV), Mouse parvovirus (MPV), Rat minute virus (RMV), Ratparvovirus (RPV) or Rat virus (RV), or a cell actively producing aparvovirus, for the preparation of pharmaceutical compositions usefulfor the treatment of brain tumors, such as glioma, medulloblastoma ormeningioma. The invention also relates to pharmaceutical compositionsfor such therapeutic applications, and to corresponding methods oftreatment of brain tumors.

[0003] 2. Description of the Related Art

[0004] Malignant human glioblastomas account for the largest number ofhuman malignant brain tumors. The conventional approaches to treatmentof gliomas include neurosurgical techniques (resection or stereotacticprocedures), radiation therapy and chemotherapy. However, despite thesetherapies glioblastomas are considered as incurable, since treatmentwith ionizing radiation, chemotherapy and/or surgical resection achievesonly a very limited prolongation of life span of patients. Typically,the average life span after diagnosis is on the order of about 12 to 16months.

[0005] It is accordingly an object of the present invention to providemeans and methods of treating brain tumors, e.g., glioma, medullobastomaand meningioma, which overcome the disadvantages of the currenttherapies and which are fundamentally different from surgicalapproaches, since even most recent surgical techniques includingneuronavigation and intraoperative MRI are not able to significantlyimprove the therapeutic outcomes for glioblastoma patients.

SUMMARY OF THE INVENTION

[0006] The present invention in one aspect relates to a method oftreating a brain tumor in a patient, such method comprisingadministering to the patient an amount of a parvotherapeutic agent thatis tumoricidally effective against such brain tumor.

[0007] The parvotherapeutic agent comprises parvovirus. Particularlypreferred species of parvovirus in the practice of the invention includeat least one agent selected from the group consisting of: parvovirus H1,LuIII, Mouse minute virus (MMV), Mouse parvovirus (MPV), Rat minutevirus (RMV), Rat parvovirus (RPV), Rat virus (RV), vectors based on theforegoing viral species, and cells capable of actively producing theforegoing viral species.

[0008] Another aspect of the invention relates to a pharmaceuticalcomposition useful for treating a brain tumor in a patient, saidcomposition comprising an amount of a parvotherapeutic agent that istumoricidally effective against said brain tumor, and a pharmaceuticallyacceptable carrier for said parvotherapeutic agent.

[0009] In another aspect, the invention relates to use of a parvovirusor a cell actively producing a parvovirus for the preparation of apharmaceutical composition for the treatment of a brain tumor.

[0010] Other aspects, features and embodiments will be more fullyapparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows a cranial MRI series for successful H-1 virustreatment of a rat, including MRI imaging before H-1 virus injectioninto the tumor (FIG. 1a), MRI imaging on day 3 after H-1 virus injectioninto the tumor (FIG. 1b) and MRI imaging on day 7 after H-1 virusinjection into the tumor (FIG. 1c). The three images in each row showdifferent sections of the same examination. The tumor is visible as awhite area in the right hemisphere (left half of the scan). The scale onthe right is 2 cm.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS THEREOF

[0012] The present invention is based on the surprising finding thatparvovirus can be successfully used for highly efficient killing ofhuman glioma cells without significantly damaging corresponding normalcells. Autonomous parvoviruses are small single-stranded DNA virusesthat rely for their replication on cellular factors expressed during theS-phase of the cell cycle. Parvoviruses are lytic viruses, i.e., theykill infected permissive cells.

[0013] In preliminary empirical work associated with the presentinvention, rat parvovirus H1, which is also infectious for humans, wasutilized. By comparing a series of established human tumor cell lines ofthe same organ origin, it was found that 5 randomly selected humanglioma cell lines (U87MG, U373MG, U138MG, U343MG and A172MG) wereparticularly susceptible to H1 virus infection compared to tumorsderived from some other organs (see Table I hereinafter). Cell survivalwas determined by clonogenicity and vital staining assays.

[0014] In addition, 6 different short term/low passage cultures frommalignant human brain tumors (5 glioblastomas and 1 gliosarcoma), whichhad been excised from patients, were propagated in vitro and furthercharacterized by the Department of Neurosurgery at the University ofHeidelberg (Germany). These also showed a high rate of susceptibility toH1 infection, similar to the above-mentioned glioma cell lines. Thehypersensitivity of human gliomas to H1 virus can be extrapolated to tworat glioma cell lines (RG2 and C6). Likewise, the murine glioma cellline G1261 was very susceptible to the related mouse virus MVMp. Theseresults are consistent with the applicability of mouse and rat gliomacells as appropriate models for the human system.

[0015] Parvotherapy according to the present invention is useful for thetherapeutic treatment of brain tumors and can significantly improve theprognosis of said tumors. Parvotherapy is usefully employed alone, oralternatively parvotherapy can be utilized in combination with othertreatment approaches, e.g., radiation, chemotherapy and/or surgicalexcision. Parvovirus H1 infection effects killing of tumor cells butdoes not harm normal brain cells. Parvotherapy can for example becarried out by intracerebral use of a suitable parvovirus, e.g.,parvovirus H1, or a related virus or vectors based on such viruses, toeffect tumor-specific therapy without adverse neurological or other sideeffects.

[0016] Thus, the present invention contemplates the use of a parvovirusor a cell actively producing a parvovirus for the preparation of apharmaceutical composition for the treatment of a brain tumor.

[0017] The term “parvovirus” as used herein comprises wild-type ormodified replication-competent derivatives thereof, as well as relatedviruses or vectors based on such viruses or derivatives. Suitableparvoviruses, derivatives, etc. as well as cells which can be used foractively producing said parvoviruses and which are useful for genetherapy, are readily determinable within the skill of the art based onthe disclosure herein, without undue empirical effort.

[0018] In one preferred embodiment of the present invention, theparvotherapeutic agents are utilized in the treatment of brain tumorssuch as glioma, medulloblastoma and/or meningioma.

[0019] In another preferred embodiment of the invention, theparvotherapeutic agent includes a parvovirus such as parvovirus H1 or arelated parvovirus such as LuIII, Mouse minute virus (MMV), Mouseparvovirus (MPV), Rat minute virus (RMV), Rat parvovirus (RPV) or Ratvirus (RV).

[0020] For administration, the parvotherapeutic agent can be combinedwith suitable pharmaceutical carriers. Suitable pharmaceutical carriersof a type well known in the art and readily commercially available,include phosphate buffered saline (PBS) solutions, water, emulsions suchas oil/water emulsions, wetting agents of various types, sterilesolutions, etc. Such carriers can be formulated with theparvotherapeutic agent(s) by conventional formulating methods foradministration to the subject at a suitable dose.

[0021] Additional pharmaceutically compatible carriers can include gels,biosorbable matrix materials, implantation elements containing thetherapeutic agent, or any other suitable vehicle, delivery or dispensingmeans or material(s).

[0022] Patients treatable by the parvotherapeutic treatment methods ofthe invention include humans as well as non-human animals. Examples ofthe latter include, without limitation, animals such as cows, sheep,pigs, horses, dogs, and cats.

[0023] Administration of the resultant parvotherapeutic pharmaceuticalcompositions to the brain tumor patient may be effected in any ofnumerous suitable ways, e.g., by intravenous, intraperetoneal,subcutaneous, intramuscular, topical, intradermal, intracranial, andintratumoral administration. The route of administration, of course,depends on the nature of the brain tumor and the specific therapeuticagent(s) contained in the pharmaceutical composition.

[0024] If such parvotherapeutic agent(s) comprise infectious virusparticles with the ability to penetrate through the blood-brain barrier,treatment can be performed or at least initiated by intravenousinjection of the viral therapeutic agent, e.g., H1 virus.

[0025] Since long-term intravenous treatment is susceptible to becominginefficient as a result of the formation of neutralizing antibodies tothe viral therapeutic agent, different modes of administration can beadopted after an initial regimen of intravenous viral administration, orsuch different administration techniques, e.g., intracranial orintratumoral virus administration, can be alternatively used throughoutthe entire course of parvoviral treatment.

[0026] As another specific administration technique, theparvotherapeutic agent (virus, vector and/or cell agent) can beadministered to the patient from a source implanted in the patient. Forexample, a catheter, e.g., of silicone or other biocompatible material,can be connected to a small subcutaneous reservoir (Rickham reservoir)installed in the patient during tumor removal or by a separateprocedure, to permit the parvotherapeutic composition to be injectedlocally at various times without further surgical intervention. Theparvovirus or derived vectors can also be injected into the tumor bystereotactic surgical techniques or by neuronavigation targetingtechniques.

[0027] Administration of the parvoviral agents or compositions can alsobe performed by continuous infusion of viral particles or fluidscontaining viral particles through implanted catheters at low flow ratesusing suitable pump systems, e.g., peristaltic infusion pumps orconvection enhanced delivery (CED) pumps.

[0028] As yet another method of administration of the parvotherapeuticcomposition is from an implanted article constructed and arranged todispense the parvotherapeutic agent to the tumoral locus. For example,wafers can be employed that have been impregnated with theparvotherapeutic composition, e.g., parvovirus H1, wherein the wafer isattached to the edges of the resection cavity at the conclusion ofsurgical tumor removal. Multiple wafers can be employed in suchtherapeutic intervention.

[0029] Cells that actively produce the parvotherapeutic agent, e.g.,parvovirus H1, or H1 vectors, can be injected into the tumor, or intothe tumoral cavity after tumor removal.

[0030] Combinations of two or more of the above-described administrationmodes can be employed in any suitable manner, e.g., concurrently,contemporaneously, or sequentially.

[0031] The dosage regimen of the parvotherapeutic agent is readilydeterminable within the skill of the art, by the attending physicianbased on patient data, observations and other clinical factors,including for example the patient's size, body surface area, age, sex,the particular virus, cell, etc. to be administered, the time and routeof administration, the tumor type and characteristics, general health ofthe patient, and other drugs or therapies to which the patient is beingsubjected.

[0032] The features and advantages of the invention will be more fullyapparent from the following non-limiting examples.

EXAMPLE 1

[0033] Parvovirus H1 Efficiently Kills Rat and Human Glioma Cells in aDose-dependent Manner

[0034] Experiments in rats showed that the intracranial injection of5×10⁷ plaque forming units (pfu) of H1 virus did not induce detectableinflammatory responses. The intracranial implantation of 3000 RG2 tumorcells caused the formation of tumors that were suppressed in adose-dependent fashion by ex vivo infection of these cells with H1virus.

[0035] Objective: The aim of this study was to evaluate the response ofrat and human glioma cells to infection with parvovirus H1 and to assessthe use of parvovirus H1 for glioma therapy.

[0036] Methods: Rat glioma cell lines C6 and RG2 and the human gliomacell line U343 were infected with different multiplicities of infection(MOI) of purified parvovirus H1. Cell growth of infected cells wascompared to growth of uninfected cells by proliferation assay (MTT-test)and by the ability of cells to form colonies after seeding the cells inculture dishes (colony forming assay).

[0037] Results: All tested cell lines were successfully infected by H1.MTT tests and colony forming assay (CFA) showed similar results, buteffects were more pronounced in the MTT test. RG2 cells were mostsusceptible to H1. After infection with an MOI of 0.05, approximately50% of the cells survived, after an MOI of 0.5 only 2% of cells remainedalive (CFA). C6 cells showed similar results after MOIs of 1 and 10(CFA). Human U343 cells reacted similarly to C6 cells. After infectionwith an MOI of 1, proliferation was reduced to 73%; at MOI 10 only 6.7%of cells survived (MTT). The results are shown in Table I below. TABLE ISusceptibility of different brain tumor cells to parovirus H1virus-induced killing Survivals at MOl 5 (%) BRAIN TUMORS Human gliomacells Cell Lines A172 10¹⁾ U87MG 0.1¹⁾ U373MG 2.7¹⁾ U343MG 18²⁾ U138MG<0.1¹⁾ Short term/low passage human glioma cells NCH37 20²⁾ NCH242 13²⁾NCH149 6²⁾ NCH89 6.9²⁾ NCH125 11.1²⁾ NCH82 2.3²⁾ Meningeoma primaryculture 101202: Survival MOI 5: 20% Medulloblastoma cell line TE671Advanced CPE after H-1 infection in cell culture. Rat glioma cell linesC6 2.4²⁾ RG2 <0.1¹⁾ Mouse glioma cell line GL261 0.4¹⁾ OTHER TUMORS 11Human Hepatoma cell lines 17-80 (Refs 1, 2)¹⁾ 3 mammary tumor cell lines25-50 (Ref 3)¹⁾

[0038] Conclusion: Parvovirus H1 is able to kill rat and human gliomacell lines with high efficacy.

EXAMPLE 2

[0039] Intracerebral Injection of Tumorsuppressive Wild-Type ParvovirusH1 does not Cause Damage to Brain Tissue in Immunocompetent Rats

[0040] Objective: The aim of this investigation was to assess whether ornot normal brain tissue exhibits signs of toxic effects after infectionwith high doses of parvovirus H1, since lack of toxicity is necessaryfor glioma therapy based on parvovirus H1.

[0041] Methods: 8 immunocompetent Wistar rats were stereotacticallyinjected with purified wild type parvovirus H1. After placing a Hamiltonneedle 4 mm into the right frontal lobe, 1.75×10⁷ particles of H1 viruswere slowly applied in a total volume of 5 μl. The animals were killed 7and 14 days after virus inoculation by lethal injection, and the brainswere fixed in 4% paraformaldehyde. Histological examination wasperformed after HE staining. Cerebrum and cerebellum were evaluatedseparately since infection with H1 during the fetal period can lead tocerebellar dysplasia in newborn rats.

[0042] Results: A total of 16 specimens that had been injected with H1were examined and compared to 2 specimens after needle placement alone.No brain section showed any signs of inflammatory response or of tissuedamage due to cell death after viral infection with H1.

[0043] Conclusion: Infection with Parvovirus H1 causes no damage tonormal cerebral and cerebellar tissue in adult rats. Since cell killingby H1 only occurred in transformed cells and H1 infection was notharmful for normal-resting cells, H1 infection was validated as a safemethodology for glioma therapy.

EXAMPLE 3

[0044] Treatment of Cerebral Tumors with Autonomous Parvoviruses(Parvovirus H-1) in a Rat Model System

[0045] Animal model: 3000 RG-2 rat glioma cells in 3 μ of DMEM culturemedium without supplements were stereotactically injected into the rightfrontal lobe of the brains of immunocompetent Wistar rats (CharlesRiver, Sulzfeld, Germany) using a stereotactic frame (David KopfInstruments, Tajunga, Calif., USA). Prior to injection of tumor cells, a4 mm long 24 gauge Abbocath-T microcatheter (Abbott Ltd., Ireland), wasplaced into a burr hole that was drilled 1 mm in front of the coronalsuture and 2 mm lateral of the midline and further into the rightfrontal lobe. The injection needle was inserted through the catheter 6mm deep. The speed of injection was 1 μl of tumor cell suspension perminute using a 10 μl-Hamilton syringe (Hamilton Company, Reno, Nev.,U.S.A.). After injection, the needle was withdrawn slowly (1 mm/min) andthe catheter was left in place. Tumors developed after 12 to 14 days.Without treatment, the lifespan of tumor-bearing animals was 18 to 21days.

[0046] Magnetic resonance imaging (MRI): Wistar rats were examined in a2.3 Tesla MRI scanner (Bruker Biospin-MRI GmbH, Tuebingen, Germany)under anaesthesia with ketamine/xylazine. Tumor formation and tumordevelopment after virus injection was monitored after intravenousinjection of contrast medium (Omniscan, Amersahm Buchler, Braunschweig,Germany). MRI scans were performed starting at day 14 after injection,every 3 or 4 days, until the animals were dead or the tumor had clearlyregressed.

[0047] H-1 virus treatment: Tumor-bearing animals were treated on days16 to 20 after injection of the tumor cells, when the formation of thetumor was demonstrated on MRI scans. The diameters of the treated tumorswere between 5 and 8 mm. After reopening of the skin, the injectionneedle of a 10 μl-Hamilton syringe containing 10 μl of purified H-1virus (titer: 5×10⁹ plaque forming units (PFU)/cell) was placed into thetumor through the microcatheter. The depth of the needle was determinedby the size of the tumor (between 8 and 10 mm). 2 μl of H-1 virus wereinjected over 2 minutes and after another minute, the needle wasretracted 1 mm. Another 2 μl of virus suspension then was injected atthe same speed (1 μl/min). This procedure was repeated a total of 5times, resulting in the administration of 10 μl of H-1 virus intodifferent locations of the tumor. In some additional animals, a secondburr hole was drilled 2 mm dorsal of the first burr hole and another 10μl of H-1 virus were injected with the same technique. In five animals,complete or near-complete regression of intracranial RG-2 gliomas wasobserved after H-1 virus treatment, including long-term survival forover 4 months in the case of two of the animals. In other animals thatwere killed 2, 4, 6 days after infection of H-1 virus into the tumor, anincreasing level of destruction of tumor cells with time after injectionwas visible. The sizes of the tumors that were treated in thisexperiment are properly considered to reflect advanced stages of tumordevelopment, leaving the untreated rats with only 3 to 6 days to live.

[0048] A series of MRI images showing the successful treatment of RG-2glioma is set out in FIG. 1. FIG. 1 shows a cranial MRI series forsuccessful H-1 virus treatment of a rat, including MRI imaging beforeH-1 virus injection into the tumor (FIG. 1a), MRI imaging on day 3 afterH-1 virus injection into the tumor (FIG. 1b) and MRI imaging on day 7after H-1 virus injection into the tumor (FIG. 1c). The three images ineach row show different sections of the same examination. The tumor isvisible as a white area in the right hemisphere (left half of the scan).The scale on the right is 2 cm.

[0049] The images of FIG. 1 indicate the effectiveness ofparvotherapeutic treatment of brain tumors in accordance with thepresent invention.

[0050] Bibliography

[0051] 1. Moehler M., Blechacz B, Weiskopf N., Zeidler M., Stremmel W.,Rommelaere J., Galle P. R., and Cornelis J. J. Effective infection, cellkilling and gene transfer of human hepatoma cells but not primaryhepatocytes by parvovirus H1. Cancer Gene Ther., 8, 158-167.

[0052] 2. Guo L. P., Li G. D., Xu H., Huang Q. S., Lin W. M., Ling W.H., Huang H., Luo Z. Y. P53 gene expression of human hepatoma cell linesand their sensitivities to parvovirus H1. Acta Biol. Exp. Sinica 1999,32, 25-29.

[0053] 3. Dupressoir T., Vanacker, J-M, Cornelis, J. J., Duponchel N.,and Rommelaere J.: Inhibition by parvovirus H-1 of the formation oftumors in nude mice and colonies in vitro by transformed human mammaryepithelial cells, Cancer Res 1989, 49, 3203-3208.

[0054] While the invention has been described herein with reference tospecific features, aspects and embodiments, it will be appreciated thatthe invention is not thus limited, but rather extends to and includesother features, aspects and embodiments, such as will suggest themselvesto those of ordinary skill in the art. Accordingly, the invention is tobe broadly interpreted and construed, as regards the spirit and scope ofthe claims hereafter set forth.

What is claimed is:
 1. A method of treating a brain tumor in a patient,said method comprising administering to said patient an amount of aparvotherapeutic agent that is tumoricidally effective against saidbrain tumor.
 2. The method of claim 1, wherein said parvotherapeuticagent comprises parvovirus.
 3. The method of claim 1, wherein saidparvotherapeutic agent comprises at least one agent selected from thegroup consisting of: parvovirus H1, LuIII, Mouse minute virus (MMV),Mouse parvovirus (MPV), Rat minute virus (RMV), Rat parvovirus (RPV),Rat virus (RV), vectors based on the foregoing viral species, and cellscapable of actively producing the foregoing viral species.
 4. The methodof claim 1, wherein said parvotherapeutic agent comprises at least oneagent selected from the group consisting of: parvovirus H1, LuIII, Mouseminute virus (MMV), Mouse parvovirus (MPV), Rat minute virus (RMV), Ratparvovirus (RPV), and Rat virus (RV).
 5. The method of claim 1, whereinsaid parvotherapeutic agent comprises parvovirus H1.
 6. The method ofclaim 1, wherein said brain tumor comprises a tumor selected from thegroup consisting of glioma, medulloblastoma and meningioma.
 7. Themethod of claim 1, wherein said brain tumor comprises a malignant humanglioblastoma.
 8. The method of claim 1, wherein said parvotherapeuticagent is administered to said patient by an administrative modalityselected from the group consisting of intravenous, intraperitoneal,subcutaneous, intramuscular, topical, intradermal, intracranial andintratumoral administration.
 9. The method of claim 1, wherein saidparvotherapeutic agent is administered to said patient by intratumoraladministration.
 10. The method of claim 1, wherein said parvotherapeuticagent is administered to said patient by intracranial administration.11. The method of claim 1, wherein said parvotherapeutic agent isadministered to said patient by intravenous administration.
 12. Themethod of claim 1, wherein said parvotherapeutic agent is administeredto said patient by administration from a source implanted in saidpatient.
 13. The method of claim 12, wherein said source implanted insaid patient comprises a subcutaneous reservoir.
 14. The method of claim1, wherein said parvotherapeutic agent is administered to said patientby injection into the brain tumor.
 15. The method of claim 14, whereinsaid injection is carried out during stereotactic surgery.
 16. Themethod of claim 14, wherein said injection is targeted byneuronavigation.
 17. The method of claim 1, wherein saidparvotherapeutic agent is administered to said patient by continuousinfusion through an implanted catheter.
 18. The method of claim 1,wherein said parvotherapeutic agent is administered to said patient byimplantation in said patient of at least one wafer impregnated with theparvotherapeutic agent.
 19. The method of claim 18, wherein theimplantation comprises implantation in a tumoral cavity formed bysurgical removal of tumor tissue.
 20. A pharmaceutical compositionuseful for treating a brain tumor in a patient, said compositioncomprising an amount of a parvotherapeutic agent that is tumoricidallyeffective against said brain tumor, and a pharmaceutically acceptablecarrier for said parvotherapeutic agent.
 21. The pharmaceuticalcomposition of claim 20, wherein said pharmaceutically acceptablecarrier is selected from the group consisting of buffered salinesolution, aqueous media, emulsions, wetting agents, and sterilesolutions.
 22. The pharmaceutical composition of claim 20, wherein saidparvotherapeutic agent comprises parvovirus.
 23. The pharmaceuticalcomposition of claim 20, wherein said parvotherapeutic agent comprisesat least one agent selected from the group consisting of: parvovirus H1,LuIII, Mouse minute virus (MMV), Mouse parvovirus (MPV), Rat minutevirus (RMV), Rat parvovirus (RPV), and Rat virus (RV).
 24. Thepharmaceutical composition of claim 20, wherein said parvotherapeuticagent comprises parvovirus H1.
 25. Use of a parvovirus or a cellactively producing a parvovirus for the preparation of a pharmaceuticalcomposition for the treatment of a brain tumor.
 26. Use according toclaim 25, wherein the brain tumor is glioma, medulloblastoma ormeningioma.
 27. Use according to claim 25, wherein the parvovirus isparvovirus H1 or a related parvovirus.
 28. Use according to claim 27,wherein the related parvovirus is LuIII, Mouse minute virus (MMV), Mouseparvovirus (MPV), Rat minute virus (RMV), Rat parvovirus (RPV) or Ratvirus (RV).